Ultrastructure and Microanalysis of Root Cementum in ...

JKAU: Med. Sci., Vol. 18 No. 4, pp: 69-82 (2011 A.D. / 1432 A.H.)

DOI: 10.4197/Med. 18-4.6

________________________________ Correspondence & reprint request to: Prof. Hisham I. Othman

P.O. Box 80209, Jeddah 21589, Saudi Arabia

Accepted for publication: 09 August 2011. Received: 17 June 2011.

69

Ultrastructure and Microanalysis of Root Cementum in

Diabetic Patients versus Healthy Patients with

Periodontitis

Suliman O. Amro, PhD, Hisham I. Othman, PhD,

and Salim M. El-Hamidy١, PhD

Department of Oral Basic and Clinical Sciences, Faculty of Dentistry and ١Department of Biological Sciences, Electron Microscopy Unit,

Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia

[email protected]

Abstract. Diabetes itself is not a direct cause of periodontal disease,

but rather it facilitates the development of gingivitis and periodontitis

through local pathological changes in the oral cavity. The relationship

between diabetes and hard dental structure, particularly root surfaces

has received far less attention, despite the fact that root surfaces are

exposed to multiple pathological factors. The aim of this study is to

evaluate the effect of diabetes type 1 and 2 on the mineralization of

periodontally diseased root cementum using scanning electron

microscope and energy dispersive spectrometry. The sample of this

study consisted of 30 periodontally diseased teeth obtained from

healthy and diabetic patients' type 1 & 2, and was classified into three

groups. The result of this study showed remarkable root cementum

destruction in diabetic group versus control group. In addition to a

significant decrease in the mineral contents, especially calcium ions in

diseased root cementum of diabetic patients' type 1, this study

concluded that the destruction of root cementum surface and the

significant decrease in the calcium contents of the cementum of

diabetic patients with periodontitis, may play an essential role of tooth

looseness in diabetic patients. Moreover, it will assist to clarify the

mechanism of periodontal destruction in diabetic patients.

Keywords: Root cementum, Diabetes, Periodontitis, Energy

dispersive X-ray analysis.

S.O. Amro et al. 70

Introduction

Several trials have demonstrated a relationship between diabetes and tooth loss

[1-3]. The status of the periodontal disease was also examined in

diabetics and a significant alveolar bone loss was observed[4]

. However, structural changes in the cementum of diabetics have not been evaluated adequately with regard to its relationship with mobility of the teeth and eventual, tooth loss. The increased frequency of tooth loss in diabetics has been associated with periodontitis, its accompanying tooth mobility, and deep pocket formation

[5]. Diabetes itself is not a direct cause of

periodontal disease, but rather it facilitates the development of gingivitis and periodontitis through local pathological changes in the oral cavity

[6].

While periodontitis in diabetics is the subject of ongoing research, the structural changes in human teeth associated with diabetes have not been adequately studied. The relationship between diabetes and hard dental structure particularly root surfaces, has received far less attention, despite the fact that root surfaces are exposed to multiple pathological factors

[7,8].

Root surface affected by periodontal disease may show various changes depending on the location of the root surface relative to the environment. When the exposed cementum comes into intimate contact with microbial dental plaque, changes occur in the diseased cementum, including hypermineralization of the cementum surface and a degeneration of the collagen matrix. In addition to a development of resorption lacunae due to penetration and/or absorption of bacterial endotoxins at the exposed cementum

[9].

Chemical analysis of the exposed cementum has shown an increase in calcium, magnesium, and phosphorus with a depth of penetration 50 um or less into the cementum. The crystals of the hypermineralized surface zone were observed to be larger than in the subjacent cementum

[10]. A limited number of studies have used an electron probe

to analyze the distribution of various elements in cementum. Hence, no consensus could be reached regarding the occurrence or, distribution of various elements

and conflicting data were reported

[11-13].

Root surfaces have been evaluated for clinical changes due to the influence of periodontal diseases. The reported results from such teeth indicated a higher Ca and P content than non-diseased root surfaces. Similarly, it has been reported that when root surfaces became exposed to the oral cavity as a result of periodontal disease, the exchange of mineral at the cementum-saliva interface resulted in a more highly mineralized

Ultrastructure and Microanalysis of Root Cementum in Diabetic Patients… 71

surface zone approximately 40 microns in depth[14]

. In the contrary to another study

[15], it was reported that exposed root structures did not

show Ca and P differences to a depth of 60 microns when evaluated by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis. They claimed that previous studies utilized preparative methods such as precipitating fixatives, embedding medium or solutions for extraction of organic matrix and dehydration, which altered the elemental content of the root surface.

Energy dispersive X-ray spectrometry (EDX) was carried out in combination with SEM. The EDX-analysis separates the x-ray spectrum by energy with enough sensitivity to show x-ray spectral data at low-beam currents. The EDX-analysis was used to determine the chemical elemental content in the diseased cementum surface

[16].

The primary composition of root cementum is of a mineralized nature, but the basic elements present, besides calcium and phosphorus, have not been verified. Opinions differ concerning the changes in cementum associated with periodontal disease. In order to understand the nature of this calcified structure in health and disease, knowledge of the elemental content of non-diseased as well as diseased root is required.

Objectives

Therefore, the present study was undertaken to evaluate the microanalysis of various elements, and assesses the surfaces characteristics of the diseased root surfaces among healthy and uncontrolled diabetic patients by using scanning electron microscopy and energy dispersive x-ray analysis.

Materials and Methods

All patients were selected from dental patients attending King Abdulaziz University, Faculty of Dentistry Male department (all male patients). Thirty hopeless vital human teeth from diabetic patients and healthy volunteers were used. Diabetic state was determined by history of previous diagnosis of diabetes. A medical history was available for each person included in the study. Local ethics committee approval was gained for the project and the informed consent was obtained from all patients. The selected patients suffered from periodontal diseases and did not receive any antibiotics or periodontal therapy during the past 6 months. All the selected teeth had periodontal pockets that ranged from

S.O. Amro et al. 72

7-9 mm probing depths and minimum 35% bone loss as determined from the radiographs. They were indicated for extraction due to advanced adult periodontitis. The collected teeth were categorized into three groups:

Group I: 10 periodontally diseased teeth from healthy volunteers (Control group).

Group II: 10 periodontally diseased teeth from diabetic patients type 1.

Group III: 10 periodontally diseased teeth from diabetic patients type 2.

During extraction, care was taken to avoid instrumentation to the areas of the root to be studied. The teeth were collected in deionized water and stored in the refrigerator. Cross root sections were cut using diamond saw at more than 5 mm apical to the cementoenamel junction. The root surface opposite the surface to be evaluated was marked with shallow groove for proper identification of the examined surface. Areas for electron microscopic examination were selected to correspond to areas examined in the EDX-analysis. All tooth samples were mounted on specimen stubs and sputtered with a 15 nm thick gold layer

§. The

specimens were then examined with a scanning electron microscope¶.

The microscope was operated at an accelerating voltage of 20 kV. The specimen was analyzed by using energy dispersive analyzer unit

║

attached the scanning electron microscope at the electron microscopic unit, Faculty of Science, King Abdulaziz University (KAU).

Statistical Analysis

Data were collected and tabulated using Microsoft Office Spreadsheet version 3.2 (Microsoft Corp., Washington DC, USA). They were also subjected to statistical analysis with ANOVA test using R (R Development Core Team, Bell Laboratories, New Jersey, USA)

[17]. The

significant level was set at 0.05.

Results

The mean age in the diabetic groups was 56.1±13.1 years versus 55±14.2 years in the control group.

_____________________ § JEOL JFC- 1600 Auto Fine Coater

¶ JSM-6360LA, JEOL, Tokyo, Japan

║EX-23000BU


Scanning Electron Microscope Examinations

Periodontally diseased cementum from diabetic patients and healthy

volunteers showed different morphological features. The periodontally

diseased root cementum of healthy volunteers (Group I), showed an

irregular, uneven surface with multiple superficial defects (Fig. 1). The

diabetic patients' type 1 with periodontally diseased teeth (Group II)

showed distinct features. The cementum surface was severely damaged

with the presence of numerous circular domes giving a pebbly

appearance with complete absence of periodontal ligament fibers. In

addition to the presence of multiple deep crack lines, also, numerous

resorption areas extended deeply into the underlying dentin (Fig. 2). In

group III, the resorption defect areas widely covered the

Fig. 1. Scanning electron micrograph of Group I (control) surface view of periodontally

diseased cementum. It appears irregular, uneven and with superficial defects (X

16000).

Fig. 2. SEM image of Group II, (type 1 DM), shows severe destruction of the cementum

surface, multiple crack lines and exposure of the underlying dentin (X 25000).

S.O. Amro et al. 74

diseased cementum surface with variable depths of penetration into the

underlying dentin, in addition to the presence of multiple crack lines

(Fig. 3).

Fig. 3. SEM image of Group III, (type 2 DM). The root surface appears widely covered by

diseased damaged cementum with the presence of multiple deep defects of variable

depths into the underlying dentin and absence of periodontal ligament fibers. (X

10000).

Electron Dispersive X-ray Analysis

The elements analyzed and compared in both groups of the energy

dispersive spectrometry study were those with an atomic number of 5 or

higher, having enough intensity that the EDX could discern them from

background or scatter radiation. The minerals most often detected in the

specimens were calcium (Ca), fluoride (F), sodium (Na), magnesium

(Mg), phosphorus (P), sulfur (S) and chloride (CL) (Table 1),

Table 1. Descriptive statistics for the different elements used in the current study.

Mean SD n Groups P value

Ca

Group I 61.75 12.89 5 G2-G1 0.04

Group II 43.72 7.16 5 G3-G1 0.56

Group II 55.09 9.04 5 G3-G2 0.21

Cl

Group I 0.46 0.34 5 G2-G1 0.07

Group II 1.54 0.93 5 G3-G1 0.17

Group III 1.31 0.67 5 G3-G2 0.85

Fl

Group I 13.08 3.80 5 G2-G1 0.46

Group II 10.73 2.76 5 G3-G1 0.46

Group III 8.72 2.31 5 G3-G2 0.56


Table 1. (Continuation) Descriptive statistics for the different elements used in the

current study.

Mean SD n Groups P value

Mg

Group I 1.68 0.35 5 G2-G1 0.61

Group II 1.42 0.17 5 G3-G1 0.93

Group III 1.77 0.62 5 G3-G2 0.41

Na

Group I 8.72 4.28 5 G2-G1 0.97

Group II 8.03 3.25 5 G3-G1 0.86

Group III 10.17 5.26 5 G3-G2 0.72

P

Group I 20.06 1.51 5 G2-G1 0.68

Group II 20.65 0.75 5 G3-G1 0.99

Group III 20.04 0.85 5 G3-G2 0.66

S

Group I 1.38 0.35 5 G2-G1 0.040

Group II 2.20 0.45 5 G3-G1 0.71

Group III 1.14 0.57 5 G3-G2 0.01

F -test p < 0.05

Group I = G1, Group II = G2, Group III = G3

In the three groups, the content of phosphorus and calcium

represents the main essential components of the diseased cementum of

healthy and diabetic patients. Calcium contents of the diseased

cementum surface of healthy controls (Group I) were higher in

comparison to the diseased cementum of diabetic patients (Group II). The

difference between them was statistically significant (p-value = 0.036)

(Fig. 4).

The influence of diabetes on calcium (Ca) contents was variable

among the two diabetic groups (II & III). The type 1 diabetic patients

showed remarkably decreased calcium in comparison to type 2 diabetic

patients, but the difference was not significant. The influence of diabetes

on fluoride (Fl) seemed to be much less dramatic since the difference

between the three groups was not significant.

The sulfur (S) contents of the diseased cementum of group II was

the highest. The difference was significant compared with controls (p

value = 0.04) and with other diabetic (Group III) (p value = 0.01)

(Fig. 5).

The difference for the other elements magnesium (Mg), sodium

(Na), chloride (Cl) and phosphate (P) among the three groups was not

significant (Table 1).

S.O. Amro et al. 76

Fig. 4. Error bars showing the mean and standard errors of calcium level in the 3 studied

groups.

Fig. 5. Error bars showing the mean and standard errors of sulfur level in the 3 studied

groups.

Discussion

This study was primarily concerned with ultrastructural and elemental changes within periodontally diseased cementum of diabetic patients. The effects of metabolic changes in diabetes are multifaceted including plaque microbiology, vascular changes, and alterations in the metabolism of collagen tissues and in immunological responses

[18].

The results of the scanning electron microscope of the diseased cementum surface of non diabetic patients revealed the presence of rough irregular surface with multiple resorption lacunae of variable depths.


This is in agreement with Adriaens et al.[19] who explained that these

lacunae may display a route of entry for bacteria into root cementum and radicular dentine. Daly et al.[20]

also showed cracks within cementum from periodontally - involved root surfaces. These surface topographical features may be of importance in therapeutic interventions aimed at rendering the root surface biologically acceptable. Eide et al.[21]

have observed a mineralized surface coating on dental cementum incident to periodontal disease. They stated that this coating is derived from components of inflammatory exudates within periodontal pockets and that this might be a reservoir of cementum-associated lipopolysaccharides.

The strongest ultrastructural destructions were observed in type 1 diabetic patients (Group II); where large areas of cemental surface were destroyed, while in type 2 diabetes the cemental destruction was mild. This is in agreement with Atar et al.[22]

who found that diabetic patients show more destructive cementum than the healthy controls. The extent of these destructions is decreasing in the order, with the least in the shorter duration of diabetes. From the SEM micrographs shown in this study, it was evident that all patients affected by a genetically determined diabetes show markedly stronger defects than the acquired diabetes. From these findings, the conclusion was drawn that genetically induced diabetes, like type 1 diabetes may lead to much more destruction than type 2 diabetes, which is normally acquired during middle-age. Oliver and Tervonen

[23] stated that, in addition to periodontitis, type 2 diabetes is

related to other complications in the oral cavity including tooth decay, dry mouth, fungal infections, as well as oral and peripheral neuropathies. The incidence of tooth loss is 15% higher in type 2 diabetic subjects compared to healthy controls. It has been suggested that this difference can be accounted for alveolar bone resorption, loss of attachment of the periodontal ligament to the cementum, and alterations in the structure and thickness of the cementum layer. The work of El-Bialy et al.[24]

unequivocally supports our suggestions concerning stronger defects in type 1 diabetes mellitus (DM). They found a decreased skeletal maturation and cephalometric measurements in diabetic patients. Their results may possibly be transferable to dental cementum, since bone tissue and cementum show similarities in their development and function as mineralized tissues.

In this study, a remarkable decrease in calcium and an increase in sulfur contents in diabetics than control were found. Other elements

S.O. Amro et al. 78

including F, Na, Mg, P, Cl, were also analyzed. No statistic difference was observed in relative content of these elements between diabetic and control. These results are consistent with the report conducted by Yurong, et al.[25]

Our findings support the hypothesis that a decrease of calcium levels

in the blood or diminished calcium incorporation into the cementum

caused by reduced cellular activity of the cementoblast may be directly

responsible for the cemental defects in diabetic patients. This is in

accordance with Balint et al.[26] who demonstrated that a glucose

concentration similar to those observed in patients with poorly controlled

diabetes causes significant inhibition of osteoblastic calcium deposition.

The recent investigation of Gunczler et al.[27] also referred to a

decreased bone mineral density and bone formation markers shortly after

diagnosis of clinical type 1 DM. It’s believed that significant loss of

calcium accounts for the marked destruction in cemental layers of

diabetic patients. Sulfur exists in many substances that are essential to

bone metabolism[28]

Therefore, obvious increase of sulfur in the current

study probably contributed to diabetic changes in the cemental tissue in

accordance with Yurong, et al.[25], who found an increase of sulfur

component in bone of diabetic patients. In this study a significant

increase in the sulfur content in type 1 diabetic patients was found. This

is in agreement with Kodaka and Debari[29]

as they reported that high

sulfur and inorganic sulfate as a result of hyperglycemia. There is no

physical explanation for an increasing sulfur value in our research, but it

could be due to an increased sulfur concentration in serum leading to its

precipitation in bone and cementum. However, elemental analysis in the

cementum of diabetic patients has not been previously evaluated and this

research may be considered as a first study concerned regarding this

point.

Further investigations on a large number of samples would be

necessary to clarify the role of elemental changes in diabetic cemental

destruction and to confirm this finding.

Conclusion

Our study concluded that the severe destruction of root cementum

surface, and the significant decrease in the calcium contents of the

cementum of diabetic patients with periodontitis, may play an essential


role of tooth looseness in diabetic patients, moreover, will assist to clarify

the mechanism of periodontal destruction in diabetic patients.

Acknowledgments

The research group acknowledges, with thanks, the Electron

Microscopy Unit, Department of Biological Sciences, Faculty of Science,

King Abdulaziz University (KAU), which assisted us during this

research. Our sincere thanks go to Dr. Mohamed El-Sehemy, professor

of oral surgery (KAU) for his support and assistance during this work.

Finally, special thanks go to Dr. Adel Abdel-Azim, professor of oral

pathology (KAU) for his valuable reviewing and editing of the research

manuscript.

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